the so-called cell density effect, which means the viral production is limited beyond a

certain cell density and cannot increase in proportion to the total cell number. This

observation is cell culture medium dependent, for example, with NSFM13 medium,

the optimum productivity of viral particles expressing GFP-Q happened when

infect the cells at 5.0E+05 cells/ml. If the cell density is doubled (infect at 1.0E+06

cells/ml), the viral production is reduced [44]. This “cell density effect” has been

observed with a number of viruses including VSV [45].

Generally, cell-culture media were designed to maximize cell growth not ne-

cessarily viral production. Consequently, a better understanding of the metabolic

limitations in the viral infection/production phase is needed to achieve high viral

productivity. Currently, two approaches have been proposed by researchers [46].

One approach is to developing feeding strategies to add critical substrates and re-

duce/remove toxic metabolites during the production phase. The second approach is

to design novel media that support not only the cell-culture growth but also the viral

production effectively which requires extensive metabolic analyses over the growth

and the production phases.

Osmolarity is also considered as a factor that significantly affects the viral yield

[19,47]. Not only the osmolarity during production but also the osmolarity during

cell growth affect the virus yield, which indicates that the history of the cells might

determine their capacity for viral production. A productive system can be achieved

by disassociating the growth phase from the production phase in terms of metabolic

and cell physiology requirements. These observations might be generalized to other

virus production systems.

11.2.2.3

Productivity of AdV in Different Operation Modes

Cultivation of mammalian cells has been realized using various technologies in-

cluding roller bottles, microcarriers, bioreactor suspension cultures, etc. Suspension

cell culture remains the most effective method for the production of AdV vectors at

large scale especially when compared to processes using adherent cells. Additionally,

with a homogeneous concentration of nutrients, metabolites, and cellular environ-

ment, the suspension culture is easy to monitor and scale-up to control process ro-

bustness and critical quality attributes of the AdV vectors. As the main cell line for

production of AdV, HEK-293 cells have been adapted to suspension culture (293S).

They were further adapted to serum-free medium 293SF, enhancing the scalability,

batch-to-batch reproducibility, and regulatory approval [48,49]. Suspension cultures

of HEK-293 in stirred tank bioreactors are projected to scale up to 10,000L, with

yields for unpurified culture in the range of 10⁹–10¹⁰ VP/ml [44,50]. Another im-

portant cell line PER.C6 has been successfully used in GMP manufacturing pro-

cesses, growing to high cell densities in serum-free suspension culture, and can be

used to produce AdV vectors in a similar fashion [50].

However, in viral production phase, the productivity of AdV vectors was limited

by cell density effect in batch mode due to the depletion of key nutrients or the

accumulation of inhibitors as previously discussed. The batch process, where cells

are exposed to a constantly changing environment, limits the growth and production

potential of the cells. Alternative modes of operation including fed-batch and

perfusion might be operated to overcome these limitations [22]. These modes of

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Bioprocessing of Viral Vaccines